We are developing a gene targeting program based on the capacity of oligonucleotides to form stable triple helix complexes with specific sequences in duplex DNA. This approach has the promise to become a simple and efficient technology for delivering DNA reactive compounds to specific sites in chromosomal DNA in living cells. Applications include gene knockout, directed gene conversion and recombination, and, perhaps, gene therapy. Triple helices have been known for over 40 years and have been the subject of many studies in vitro. However there is direct evidence that the structure of mammalian chromatin would preclude access to triplex forming oligos (TFO). We prepared a TFO, linked to a photoactivatable DNA mutagen, directed against a sequence in a gene (HPRT) frequently used as a mutation reporter. We introduced this into mammalian cells and, after photoactivation of the mutagen, isolated colonies of cells with mutations in the target gene. Sequence analysis showed that the mutations were located at the target sequence within the gene. We have prepared TFOs with novel sugar modifications that show enhanced targeting activity. Treatment of S phase cells with these TFOs results in 30% of targeted crosslinking and 5-10% mutation frequencies. Both crosslinking and mutagenesis are much lower in quiescent cells. These results indicate that the accessibility of chromosomal target sites in mammalian cells is modulated by the biology of the cell. Furthermore the frequency of mutagenesis is sufficiently high to allow identification of colonies with sequence changes in simple screens of a few clones. We also find that the targeting oligonucleotides can be used to direct homologous recombination. Cells treated with the TFO and a donor DNA with homology to the region around the target sequence show knock in of the donor DNA at frequencies 3-4 orders of magnitude above that seen with cells treated with donor alone.